Previous studies from our laboratory have demonstrated that when NK cells expressing both activating and inhibitory Ly49 receptors are presented with a ligand, inhibition dominates unless co-stimulation with IL-12 or IL-18 is present. Overriding the inhibitory Ly49G2 receptor blockade for cytokine production was observed both in vitro and in vivo. In the present study, we analyzed the regulation of other ITAM positive receptors on NK, NKT and T cells. Our studies demonstrate that crosslinking of NKG2D and PK136 results in a synergistic NK response when combined with IL-12 or IL-18. Examination of NKT and T cells demonstrated that cross-linking of NKG2D and CD3 resulted in potent synergy when combined with IL-12, and to a lesser degree with IL-18. Other IL-12 family members, IL-23 and IL-27, also demonstrated a similar co-activation signal with ITAM bearing receptors, albeit to a lesser degree. Similar to our previous Ly49D report, ITAM bearing receptor synergy was most evident by production of IFN-g, as an endpoint; other cytokines including TNFa and IL-2 were not similarly effected. Mechanistic examination of the synergy indicated a potent regulation of mRNA that was evident in both the nuclear and cytoplasmic compartment and involved both processed and unprocessed mRNA. In addition, our studies demonstrated that as little as 15 minutes of IL-12 pretreatment was sufficient to result in maximal activation in synergy with activating Ly49s. This synergy could be even further enhanced by combinations of sub-optimal doses of IL-12 and IL-18 with activating Ly49s and could be observed in vivo with NKG2D. Finally, the IL-12 synergy with ITAM bearing receptors appeared to be dependant on STAT4 activation since STAT4-KO mice failed to demonstrate this potent synergy. Thus, our data demonstrates the importance of multiple signals for maximizing the innate immune response.NKT and NK cells are important immune regulatory cells. The only efficient means to selectively stimulate NKT cells in vivo is alpha-galactosylceramide (alphaGalCer). However, alphaGalCer effectively stimulates and then diminishes the number of detectable NKT cells. It also exhibits a potent, indirect ability to activate NK cells. We have now discovered another ceramide compound, beta-galactosylceramide (betaGalCer) (C12), that efficiently diminishes the number of detectable mouse NKT cells in vivo without inducing significant cytokine expression or activation of NK cells. Binding studies using CD1d tetramers loaded with betaGalCer (C12) demonstrated significant but lower intensity binding to NKT cells when compared with alphaGalCer, but both ceramides were equally efficient in reducing the number of NKT cells. However, betaGalCer (C12), in contrast to alphaGalCer, failed to increase NK cell size, number, and cytolytic activity. Also in contrast to alphaGalCer, betaGalCer (C12) is a poor inducer of IFN-gamma, TNF-alpha, GM-CSF, and IL-4 gene expression. These qualitative differences in NKT perturbation/NK activation have important implications for delineating the unique in vivo roles of NKT vs NK cells. Thus, alphaGalCer (which triggers NKT cells and activates NK cells) efficiently increases the resistance to allogeneic bone marrow transplantation while betaGalCer (C12) (which triggers NKT cells but does not activate NK cells) fails to enhance bone marrow graft rejection. Our results show betaGalCer (C12) can effectively discriminate between NKT- and NK-mediated responses in vivo. These results indicate the use of different TCR-binding ceramides can provide a unique approach for understanding the intricate immunoregulatory contributions of these two cell types.